Project Summary/Abstract HIV and HTLV are both retroviruses that cause life-threatening, incurable disease in millions of individuals throughout the world. These retroviruses insert their DNA into the genomes of the cells they infect. A fraction of the the infected cells do not produce virus. There is little, other than the proviral DNA, that distinguishes these latently-infected cells from those that have not been infected. In contrast to current therapeutics, which target viral proteins (i.e. HIV protease inhibitors or reverse transcriptase inhibitors), we are developing a new class of retroviral therapeutics that target the proviral DNA itself. Our approach uses engineered recombinases that specifically act on the Long Terminal Repeats (LTRs) that flank the integrated provirus. We have already demonstrated that such recombinases can efficiently remove HIV provirus from patient-derived cells when delivered using a lentiviral vector. We have also shown that there are no deleterious effects when the engineered, anti- HIV recombinase is constitutively expressed in transgenic mice. In contrast to CRISPR/Cas9 and other nuclease-based approaches to targeting proviral DNA, recombinases act with single-nucleotide precision thereby making them intrinsically better for this purpose than nucleases. (The unpredictable indels associated with anti-HIV CRISPR/Cas9 appear to enhance the rate of viral escape.) This proposal targets the primary limitation of the engineered recombinase approach, namely that altering recombinase specificity is extremely time consuming. To date, engineering of new recombinases has relied on random mutagenesis as a source of genetic diversity. This is highly inefficient since the overwhelming majority of mutations screened occur in regions of the protein that are not associated with DNA recognition. Moreover, given the vast number of potential mutations across the entire protein, random screening, necessarily, samples the available sequence-space very sparsely. In this application, we propose to use a new, structure-guided approach to identifying recombinases with altered specificity. We will again screen millions of clones for the desired activity, but the genetic diversity in our screens will be limited to key regions of the protein, and only a sensible subset of the amino acids at these key positions will be part of the library. (To clarify precisely which amino acids should be varied, we will determine crystal structures of anti-HIV and anti-HTLV recombinases in complex with their LTR targets and probe the functional role of specific mutation sites within the recombinases.) We anticipate that dense-sampling of the most relevant regions of sequence-space will dramatically streamline the recombinase design process. This will allow us to quickly target HIV and HTLV strains that our current-best recombinases cannot excise.